Fig 1.
An. coluzzii and An. gambiae females were infected with P. falciparum field isolates following EcR-silencing.
(A-B) An. coluzzii and An. gambiae females were injected with either dsGFP (Cntrl) or dsEcR and then infected with P. falciparum field isolates originating from six different gametocyte carriers (designated patient, p#). (A) Two batches of An. coluzzii females were infected with parasites from p1, p2, and p3. (B) Three batches of An. gambiae females were infected with parasites from p1, p3, p4, p5, and p6. (C) In An. coluzzii, dsEcR-injections did not reduce cumulative egg development to the level of statistical significance (Mann-Whitney). (D) In An. gambiae, EcR-silencing did result in a significant reduction in cumulative egg numbers compared to controls (unpaired t-test). (E-F) In both species, (E) An. coluzzii and (F) An. gambiae, dsEcR-injections had no effect on cumulative oocyst prevalence (Fisher’s Exact) or intensity (unpaired t-test and Mann-Whitney). P next to pie charts = prevalence. N = sample size. p# = parasite isolate.
Fig 2.
Oocyst growth is accelerated after dsEcR treatment in both An. coluzzii and An. gambiae.
(A) In An. coluzzii and (B) An. gambiae, EcR-depleted females had significantly larger oocysts at 8 days pIBM compared to dsGFP controls (Cntrl) (GLMM, LRT). Average oocyst size per midgut is shown for simplicity–analyses are based on nested data incorporating all oocyst measurements. (C) Representative images of oocysts found in the midgut of An. gambiae control and dsEcR females. Black dotted circles show oocyst perimeters. Scale bar = 100 μm. (D) At 12 days pIBM, EcR-silenced An. gambiae females had a greater number of sporozoites in their salivary glands than controls (Mann-Whitney). Sporozoite prevalence (P) at this time point was not different (Fisher’s Exact). (E) Expression of Lipophorin in dsEcR females was elevated relative to dsGFP controls (unpaired t-test). Gene expression was assessed for each batch of mosquitoes used in infections (whole body mosquito sample without head, prior to blood feeding). For applicable panels, N = sample size, or number of mosquitoes. Noocysts = number of individual oocyst measurements. p# = parasite isolate.
Fig 3.
P. falciparum oocyst growth is negatively linked to egg development.
(A) In An. coluzzii controls (dsGFP-injected), egg numbers are negatively associated with oocyst size, but this association is lost following dsEcR treatment (GLMM, LRT). (B) In An. gambiae, egg numbers are negatively associated with oocyst size in both control and dsEcR conditions (GLMM, LRT), but (C) this association differentially varies across oocyst density in control and dsEcR females (3-way interaction, treatment*egg#*oocyst#, GLMM, LRT, X21 = 8.57, p = 0.003). Lines across egg numbers and oocyst size graphically represent the model-based analysis that was performed, which used nested individual oocyst measurements. Shading shows 95% confidence interval. N = sample size, or number of mosquitoes. Number of individual oocyst measurements including in analysis were: An. coluzzii controls = 926, An. coluzzii dsEcR = 1003, An. gambiae controls = 669, An. gambiae dsEcR = 760.
Fig 4.
Individual P. falciparum isolates vary in their growth response to EcR-silencing.
(A) For An. coluzzii females, both p1 and p2 parasites produced larger oocysts in EcR-silenced females, but p3 parasites failed to respond to dsEcR conditions and did not grow larger than controls (GLMM, LRT). (B) For An. gambiae, all P. falciparum isolates responded to EcR-silencing by growing larger except for p6 parasites, which remained the same size as controls (Cntrl), even though they were fed to the same batch of mosquitoes as p5 parasites (GLMM, LRT). (A-B) Oocyst size does not vary significantly between parasite isolates in dsGFP controls for An. gambiae or An. coluzzii (An. coluzzii dsGFP: GLMM, LRT X22 = 1.52, p = 0.466; An. gambiae dsGFP: GLMM, LRT X24 = 3.47, p = 0.483), yet it does for dsEcR-treated females in both species (An. coluzzii dsEcR: GLMM, LRT X21 = 6.3, p = 0.012; An. gambiae dsEcR: GLMM, LRT X21 = 4.67, p = 0.031). (C) Oocyst size varies significantly between An. coluzzii and An. gambiae control females infected with the same parasite isolates (p1 and p3) (GLMM, LRT), whereby oocysts are larger in An. coluzzii than in An. gambiae. (D) An. coluzzii were collected as larvae from VK5, Burkina Faso and infected with P. falciparum isolates p1-p6. Pre-gravid females that failed to develop eggs were less likely to become infected (pie charts, P = oocyst prevalence, Fisher’s Exact), although among infected individuals, there was no difference in oocyst intensity (Mann-Whitney). (E) Pre-gravid females had significantly larger oocysts at 7 days pIBM compared to gravid females (GLMM, LRT). For all applicable panels, average oocyst size per midgut is shown for simplicity–analyses are based on nested data incorporating all oocyst measurements. N = sample size, or number of mosquitoes. Noocysts = number of individual oocyst measurements. p# = parasite isolate.
Table 1.
Gametocytemia and COI for P. falciparum field isolates.
P. falciparum parasites were collected from six different gametocyte carriers (p1-p6 and used for infections in An. coluzzii and An. gambiae females. Prior to infection, a blood smear was used to count the number of gametocytes present in each sample. Gametocytes were counted per 1,000 leukocytes and then, using an estimated conversion factor of 8,000 leukocytes per μl of blood, converted to gametocytes per μl of blood (Gams/μl). MSP1-typing was performed on dried blood spots or recently thawed frozen blood samples and used to determine the (minimum) number of unique parasite genotypes, or complexity of infection (COI), present in each isolate.